The following cell lines were used in the present study: Pfeiffer and Toledo, which are diffuse large B cell lymphoma (DLBCL) cells, and Hut78, which corresponds to a cutaneous T-cell lymphoma (CTCL). Cells were obtained from the American Type Culture Collection (ATCC; Manassas VA, USA). The Pfeiffer cell line carries the A677G mutation in EZH2, while Toledo cells are wild-type. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂ in complete medium, comprising RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution (all from Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Ribavirin was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany); 3-deazaneplanocin A (DZNep) served as the control and was purchased from Calbiochem (cat. no. 120964-45-6). Ribavirin and DZNep were dissolved in RPMI-1640 medium, stored at −20°C and thawed before use. Stock solutions were thawed/frozen no more than three times. Cell lines were treated with 10, 15, 20, 25 and 50 µM of ribavirin, or 0.1 µM DZNep for 24, 48 and 120 h.

Cells were seeded in 25-cm² culture flasks (Corning Inc., Corning, NY, USA) at a density of 2×10⁵ cells in 5 ml of complete medium. Cells were then treated with ribavirin at the indicated concentrations; the medium containing the drug was replaced daily. DZNep at a concentration of 0.1 µM was used as a positive control. After 24, 48 and 120 h, cells were stained with 0.4% trypan blue to assess cell viability and counted using a TC10 Automated Cell Counter (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All assays were performed in triplicate. The viability of cells under each treatment condition was expressed as a percentage of cell count relative to the untreated control cells.

Exponentially proliferating cells (2×10⁵) were plated in a 25-cm² cell culture flask and incubated in RPMI-1640 medium and treated with ribavirin (10, 25 and 50 µM) or DZNep (0.1 µM) for 120 h. Untreated cells served as the negative control. After 120 h of treatment, the cells were collected; 2×10³ cells were seeded into 25-cm² culture flasks for ≤16 days in complete drug-free medium. The medium was discarded every 48 h and replaced with complete fresh medium. Cells were cultured for 16 days, after which the viability of cells was determined by a trypan blue exclusion assay using a TC10 Automated Cell Counter (Bio-Rad Laboratories, Inc.).

Three independent experiments in triplicate were performed, and data are expressed as the mean ± standard deviation. Data were statistically analyzed using GraphPad Prism V6 software (GraphPad Software Inc., La Jolla, CA, USA). Significant differences were determined using one-way analysis of variance (ANOVA) followed by Bonferroni correction to determine significant differences between each experimental group against its respective control. P<0.05 was considered to indicate a statistically significant difference.

Hut78 cells (2.5×10⁵) were cultured in 25-cm² flasks and treated with ribavirin for 120 h. Once cells were washed with PBS and centrifuged for 120 × g for 5 min, proteins were extracted using radioimmunoprecipitation buffer (150 mM NaCl; 1.0% IGEPAL CA630; 0.5% sodium deoxycholate; 0.1% SDS and 50 mM Tris, pH 8.0) in the presence of proteinase inhibitors (cat. no. p8340; Sigma-Aldrich; Merck KGaA). The protein concentration was determined using a bicinchoninic acid assay (BCA-1, Bio-Rad Laboratories, Inc.) and the integrity was assessed by Coomassie staining. A total of 30 µg protein was separated by 10% SDS PAGE and transferred onto a polyvinylidene difluoride membrane (cat. no. 1620177; Bio-Rad Laboratories, Inc.). The membrane was blocked with 5% skim milk in PBS for 1 h at room temperature and subsequently incubated with antibodies against EZH2 (cat. no. 36-6300; 1:500, Invitrogen; Thermo Fisher Scientific, Inc.), STAT-1 (cat. no. sc417; 1:200, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and SCD (cat. no. sc58420; 1:500,) (Santa Cruz Biotechnology, Inc.), and anti-actin peroxidase (cat. no. A3854; 1:10,000, Sigma Aldrich; Merck KGaA) in blocking solution (5% skim milk in TBS + 0.1% Tween-20), overnight at 4°C. The following secondary antibodies were used: for EZH2, anti-rabbit (cat. no. sc2370) and for STAT-1 and SCD, anti-mouse (cat. no. sc2371), which were obtained from Santa Cruz Biotechnology, Inc. The secondary antibodies were diluted 1:1,000 and the incubation was performed for 1 h at room temperature. Protein bands were visualized using Clarity Western Enhanced Chemiluminescence Substrate (cat. no. 1705060; Bio Rad Laboratories, Inc.). Bands were quantified densitometrically using ImageJ version 1.50f (National Institutes of Health, Bethesda, MD, USA).

Histone proteins were isolated via the sulfuric acid extraction method. Briefly, the nuclear pellet was resuspended in 0.4 M H₂SO₄ for 4 h; following centrifugation (10,000 × g, 20 min), acid-soluble proteins in the supernatant were obtained via overnight precipitation with 20% trichloroacetic acid and centrifugation (16,000 × g for 30 min). The pellets containing histone proteins were washed once in ice-cold acetone containing 1% HCl and followed by ice-cold acetone alone. The pellet was dried under vacuum and stored at −80°C. The protein concentration was determined by the Bradford method, and the integrity of the histone proteins in the acid-soluble extract was evaluated using Coomassie staining. Proteins were separated via 15% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.). The membrane was incubated for 1 h with blocking solution (TBS-Tween-20, 5% of non-fat milk) followed by overnight incubation with antibodies, including anti-H3K27me3 (cat. no. 07-449) and anti-H3 total (cat. no. 06-755) obtained from EMD Millipore (Billerica, MA, USA). Anti-rabbit secondary antibody (cat. no. sc2370; 1:1,000, Santa Cruz Biotechnology, Inc) was then applied for 1 h at room temperature. Protein bands were visualized using the chromogenic substrate Clarity Western Enhanced Chemiluminescence Substrate (cat. no. 1705060, Bio-Rad Laboratories, Inc.). Bands were quantified densitometrically using ImageJ software.

Hut78 cells were treated with 50 µM ribavirin for 120 h as aforementioned and then total RNA was isolated using TRIzol (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. RNA quality was evaluated by capillary electrophoresis (Agilent 2100 Bioanalyzer, Agilent Technologies, Inc., Santa Clara, CA, USA); only RNA samples with an RNA integrity number >8.0 were further processed for microarray analysis. A total of 200 ng RNA from each experimental cell group was evaluated using the Gene Chip Human Transcriptome Array 2.0 (Affymetrix; Thermo Fisher Scientific, Inc.) to determine the whole transcriptome expression profiles according to the manufacturer's protocols. Briefly, the synthesis and amplification of cDNA, and gene expression profiling were conducted using the WT PLUS Reagent Kit for fresh samples (Affymetrix; Thermo Fisher Scientific, Inc.). Washing and staining of the samples were performed using the Gene chip hybridization wash and stain kit in the Gene Chip Fluidics Station 450 system (Affymetrix; Thermo Fisher Scientific, Inc.). The probe arrays were scanned using The Gene Chip Scanner 30007G (Affymetrix; Thermo Fisher Scientific, Inc.). Signal intensities of the array were analyzed with Affymetrix Expression Console software (version 1.3). Briefly, raw data probes were normalized using Signal Space Transformation-Robust Multichip Analysis for background correction and to obtain the quantile algorithm. To define the differential expression profiles of the different conditions, two-way ANOVA was performed in the Affymetrix Transcriptome Analysis Console software (version 3.0). Genes with a fold change >2 or <2 and with an P≤0.05 (obtained via ANOVA) were considered significantly altered between the conditions. All data were uploaded in Gene Expression Omnibus: GSE118866, token: Klujuowqpnsvpkl.

Gene Ontology analysis was performed using Protein Analysis Through Evolutionary Relationship (http://www.pantherdb.org). Molecular pathways and interaction networks were analyzed by Ingenuity Pathway Analysis (Ingenuity Systems Inc., Redwood City, CA, USA).

Hut78 cells were treated with ribavirin for 120 h, and total RNA was isolated when cells attained ~70% confluence, using TRIzol reagent according to the manufacturer's protocols. RNA purity and integrity were assessed via spectrophotometric analysis using a NanoDrop 2000c spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA) and denaturing 2% agarose gel. Bands were visualized using a MiniBIS Pro D-Transilluminator (DNR Bio-Imaging Systems Ltd., Neve Yamin, Israel). A total of 1 µg total RNA was used for cDNA synthesis with the GeneAmp RNA PCR Core kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). iQ SYBR Green SuperMix (Bio-Rad Laboratories, Inc.) was used according to the manufacturer's protocols. qPCR reactions were run in triplicate using an ABI PRISM 7000 (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions for qPCR were as follows: 10 min at 95°C; 40 cycles of 30 sec at 95°C and 30 sec at 60°C. Data were analyzed using the 2^−ΔΔCq method (12), and reported as the fold-change in gene expression normalized to the endogenous control gene hypoxanthine phosphoribosyltransferase 1 (HPRT1), and relative to untreated cells. The primers used were: HPRT1 forward, 5′-GAACCTCTCGGCTTTCCCG-3′ and reverse, 3′-CACTAATCACGACGCCAGGG-5′; STAT-1 forward, 5′-ATGCTGGCACCAGAACGAAT-3′ and reverse, 3′-GCTGGCTGACGTTGGAGATC-5′; and SCD forward 5′-GGGATCCTTCAGCACAGGAA-3′ and reverse 3′-CACCGCTTCTCCAATGGATT-5′. Annealing temperatures were 60°C for all reactions. Three independent triplicates were conducted. P-values were calculated using a two-tailed t-test.

Hut78 cells were plated (2×10⁵) in a 6-well plate 24 h prior to viral infection with 1 ml of complete optimal medium (with serum and antibiotics) and were incubated overnight at 37°C and 5% CO₂. Then, the medium was removed from the wells and replaced with 1 ml of complete medium with Polybrene (cat. no. sc-134220, Santa Cruz Biotechnology, Inc.) at a final concentration of 2 µg/ml. Lentiviral particles were thawed at room temperature and mixed gently before use. Subsequently, cells were infected by adding STAT-1 shRNA Lentiviral Particles (cat. no. sc-44123V, Santa Cruz Biotechnology, Inc.) to the culture and centrifuged at 2,460 × g for 1 h, and then mixed for 6 h at 37°C in 5% CO₂. Control shRNA Lentiviral Particles (cat. no. sc-108080, Santa Cruz Biotechnology, Inc.) were used. The next day, the culture medium was removed and replaced with 1 ml of complete medium (without Polybrene) and cells were incubated overnight at 37°C in 5% CO₂. Then, the shRNA Lentiviral Particles were diluted 1:3 according to the manufacturer's instructions and added to the cells; the cells were incubated for 24–48 h in complete medium. The clones expressing the shRNA were selected with puromycin, and the medium was replaced with fresh puromycin-containing medium every 3–4 days until resistant cells could be identified. Cell viability assays were performed using cells treated with 50 µM ribavirin for 120 h as aforementioned. The experiments were conducted with at least three independent triplicates. P-values were calculated using one-way ANOVA with Bonferroni correction.

Lymphoma cells were exposed to different concentrations of ribavirin and the cell viability was analyzed at 120 h post-treatment. As presented in Fig. 1A-C, a dose-dependent effect of different extents was observed in the cell lines. For Pfeiffer cells, no inhibition at 24 and 48 h was reported regardless of the dose of ribavirin administered; however, a significant effect was observed with the three concentrations at 120 h. Inhibition with DZNep revealed a significant effect at 120 h. For Toledo cells, suppressed cell viability was observed following treatment with 25 µM ribavirin for 120 h, and with 50 µM ribavirin at 24 and 120 h. DZNep treatment also revealed a significant effect on cell viability at 48 and 120 h. In the CTCL cell line Hut78, significant inhibition was observed with 25 µM ribavirin at 48 h and 120 h as well while 50 µM exhibited inhibitory effects at 24, 48 and 120 h. Of note, this cell line appeared to be the most sensitive to ribavirin, but less so to DZNep.

A clonogenic assay was conducted to determine the colony formation ability of cells. The results suggested that the colony formation ability of Pfeiffer cells was significantly inhibited with all doses of ribavirin. On the contrary, the colony formation ability of Toledo cells was markedly unaffected in response to ribavirin; however, significant inhibition was observed following treatment with DZNep. In the CTCL cell line Hut78, a significant decrease in clonogenicity was observed in response to 50 µM ribavirin, while DZNep did not change the clonogenicity of this cell line (Fig. 2A-C).

To determine whether the effects of ribavirin on the viability and clonogenicity of the lymphoma cell lines were associated with EZH2 and H3K27m3, cells were treated for 72 h with the aforementioned doses of ribavirin and DZNep. The results indicated that the expression of EZH2 was markedly unaffected in response to ribavirin in the cell lines, whereas a notable reduction in Pfeiffer cells was observed following treatment with DZNep (Fig. 3A). Of note, no marked reductions in H3K27 trimethylation were reported in response to ribavirin (Fig. 3B).

A total of 978 genes were reported to be differentially expressed. Among these, 629 were upregulated and 349 were downregulated. Ingenuity Pathway Analysis of the microarray results (Table I) revealed that KLHDC7B, PTGS2, GBP1, STAT1, GBP5, GBP2, GBP4, WARS, RGS1 and GBP1P1 were the top 10 most upregulated genes, while SCD, TIMP2, GIPC3, DUSP9, CD5, TCF7, RFLNB, SBK1, LGMN and SMIM24 were the most downregulated. The top canonical pathways most significantly affected by ribavirin treatment included ‘antigen presentation’, ‘communication between innate and adaptive immune cells’, ‘cross-talk between dendritic and natural killer cells’, ‘unfolded protein response’ and ‘allograft rejection’. The top regulators of these pathways were determined to be STAT1, IFN-γ, RELA, IFN-α and IFN-α2.

STAT1 and SCD, which were determined to be upregulated and downregulated, respectively, were analyzed by RT-qPCR and western blotting. The results demonstrated that in both cases, STAT1 expression was increased, while that of SCD was decreased at the RNA and protein levels; these differences were statistically significant (Fig. 4A-D). As STAT1 was proposed as a top regulator of the central canonical pathways determined to be altered in microarray analysis, this gene was deleted using shRNA. As presented in Fig. 5, an ~70% reduction in cell viability was observed following STAT1-shRNA Lentiviral Particles knockout; however, cell viability was markedly unaffected in response to the scramble control miRNA. Treatment with ribavirin of STAT1-depleted cells further decreased cell viability; these differences were statistically significant.